Chemical composition and vascular and intestinal smooth muscle relaxant effects of the essential oil fromPsidium guajavafruit

Context:Psidium guajava L. (Myrtaceae) is widely used in traditional medicine for the treatment of various ailments including cardiovascular and gastrointestinal disorders. Objectives: The current study investigated the chemical composition and cardiovascular and gastrointestinal effects of the essential oil of P. guajava. Materials and methods: The chemical composition of the essential oil was investigated using gas chromatography-mass spectrometry (GC-MS) technique. The biological activity of the essential oil was tested on rabbit aorta and jejunum. All changes in isometric tension were recorded through a force transducer coupled with a bridge amplifier data acquisition system. Results and discussion: GC-MS analysis showed the presence of butanoic acid methyl ester, 3-methyl glutaric anhydride, 1-butanol, 3-hexenal, cinnamyl alcohol, 1-hexanol and hexane as the major components. In isolated rabbit aorta preparations, the essential oil showed vasorelaxation at doses of 3-10 mg/mL against high K⁺ and phenylephrine pre-contractions with EC₅₀ values of 5.52 (5–6.04) and 6.23 mg/mL (5.0–7.46). The essential oil inhibited spontaneous and high K⁺ induced contractions in isolated rabbit jejunum with EC₅₀ values of 0.84 (0.3–1.38) and 0.71 mg/mL (0.3–1.12) and shifted Ca ⁺² concentration curves to the right, similar to verapamil, suggesting spasmolytic activity mediated possibly through Ca ⁺² channel blockade. Conclusions: In summary, the data indicated the presence of seven different phytoconstituents in the essential oil of P. guajava and calcium channel blocking activity, which provides a pharmacological base to the traditional use of P. guajava in cardiovascular and gastrointestinal disorders. Further studies are suggested to explore the molecular nature of these effects.

Keywords: Guava fruit; Myrtaceae; spasmolytic; vasorelaxant

Psidium guajava L. (Myrtaceae), commonly known as "Guava", is native to Central and South America and found in tropical and subtropical countries, including Pakistan (Alves et al. [1]). It ranks fourth after mango, citrus and bananas on the basis of production in Pakistan (Anka [2]). The Guava fruit is commercially important due to its flavour and aroma, while nutritionally it is an excellent source of vitamin C, niacin, riboflavin and vitamin A (Khadhri et al. [17]). The bark of the plant is used to treat diarrhoea, dysentery and upper respiratory tract infections (Burkill et al. [5]). Leaves are used for treating cough, pulmonary disorders, diarrhoea, vomiting, gastroenteritis, spasms, wounds and ulcers (Ticzon [33]; Lutterodt et al. [19]; Ross [24]; Shen et al. [30]). The fruit is tonic, cooling, laxative and anthelmintic (Ticzon [33]), while the flowers and leaves possess antibiotic activity (Watt & Breyer-Brandwijk [34]). Similarly, extracts from various parts of P. guajava are used in the treatment of wounds, ulcers, toothache, cough, sore throat, inflamed gums and other diseases (Morton [21]).

The essential oil obtained from Guava fruit contained about 21 compounds, which include p-caryophyllene (23.8%), α-copaene (2.7%) and α-humulene (3.1%) as the major constituents. The oil also contained (Z)-p-ocimene, (E)-p-ocimeneareand limonene. p-caryophyllene, α-humulene and α-selinene contribute significantly to Guava flavour (Macleod & De Troconis [20]). Moreover, leaf extract contains β-caryophyllene, β-selinene, α-selinene, β-caryophylla-4,8-globulol, trimethyl cyclododecanodiol and dioctyl phthalate (Silva et al. [31]). The chemical constituents found in different parts of plants vary from region to region. Guava fruit has a reputation in the management of cardiovascular and gastrotintestinal disorders in traditional medicine (Goh et al. [12]; Burkill et al. [5]; Ticzon [33]; Yamashiro et al. [35]); however, its fruit has been ignored in the past for scientific studies in this regard. Therefore, in the current study, we investigated the chemical composition of oil obtained from guava fruit using GC-MS analysis and its effect on vascular and intestinal smooth muscles preparations.

The chemicals and standard drugs acetylcholine perchlorate, verapamil hydrochloride, potassium chloride, calcium chloride, dimethyl sulphoxide (DMSO) and ethylenediamine tetraacetic acid (EDTA) were obtained from Sigma Chemical Co. (St Louis, MO). Calcium chloride and sodium sulphate were acquired from Merck (Merck, Darmstadt, Germany). Chloroform was purchased from Lab-Scan Company Ltd. (Bangkok, Thailand). Diethyl ether was purchased from Reanal Fine Chemicals Co. (Hungary). The chemicals and drugs were dissolved in distilled water or DMSO according to the relevant protocol. All chemicals were of analytical grade.

The fresh edible fruit of P. guajava (10 kg) was purchased from a local market and stored at room temperature. The Guava fruit was identified and authenticated by Dr. Shazia Anjum, Director, Cholistan Institute of Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan. The specimen was deposited in the herbarium of the same institute with a voucher specimen number 3518/CIDS/IUB.

Steam distillation was employed to obtain the essential oils from the Guava fruit. The distillation process was performed 20 times for 90 min each. Steam was produced in a generator and passed through a glass pipe to the fresh fruit. The vapours produced were passed through a condenser and collected in the receiver as a distillate. The distillate was shaken with diethyl ether in a separating funnel and the mixture was allowed to stand for 1 h. The organic layer containing the essential oil was collected and the water traces were removed by treating with sodium sulphate. The organic layer was finally evaporated at ambient temperature to get pure essential oil (Rasheed et al. [23]).

The essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS) (PerkinElmer, Waltham, MA). GC analysis was carried out on a Clarus-600, Elite-5 MS model equipped with an Elite FFAP, 250 μm (internal diameter) and 30 m (length) column. A flame ionization detector was used. A Clarus-600-C mass spectrometer with an electron ionization detector was used. The injection temperature was 250 °C while GC temperature was started at 50 °C and increased at the rate of 10 °C/min to 300 °C (end temperature). The sample was run at a rate of 1 mL/min and Helium was used as the carrier gas. The injection volume was prepared in chloroform. The mass spectrum was obtained at 70 eV with a mass scan range of 0–450 amu. The mass spectrum was compared with a fragmentation pattern available at NIST Library (Paniandy et al. [22]).

In the current study, male rabbits of local breed were used as the model animal. Animals were used and cared for according to the rules of the ethical committee of COMSATS Institute of Information Technology, in compliance with the recommendations of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (NRC, 1996). All the animals were kept at temperature of 23–25 °C and were given free access to food and water. The rabbits were kept on starvation for 24 h before experimentation.

The vascular reactivity studies of the essential oil were carried out on rabbit aortic rings as reported previously (Shah & Gilani [28]). The animals were euthanized by cervical dislocation; the aorta was removed from the thoracic and cut in to 2–3 mm pieces. The rings were suspended individually between two stainless steel hooks. One hook was attached to a steel rod at the bottom of the organ bath and the second one was attached to a force transducer (MLT 0201), continuously bubbled with 5% CO2 in O2 (carbogen) in normal Kreb's solution maintained at 37 °C. Kreb's solution was composed of (mM): NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4 1.3, MgSO4 1.2 and glucose 11.7 (pH 7.4). A preload of 2 g was used for individual tissue and the tissue preparations were kept uninterrupted for a 1 h equilibrium period before applying test samples. K+ (80 mM) and phenylephrine (PE 1 μM) solutions were used to stabilize the preparations. The effects of the essential oil were determined against the PE and K+ (80 mM)-induced contractions. Variations in the isometric tension of the aorta preparations were assessed with the help of a force–displacement transducer attached to a Power Lab data acquisition system (AD Instruments, Sydney, Australia).

The smooth muscle relaxation or contraction activities of the essential oil were performed on an isolated rabbit jejunum preparation, as described previously (Shah et al. [27]). The animals were sacrificed by cervical dislocation, abdomen was cut open and the jejunum was isolated. Isolated jejunum preparations of 2–3 cm were freed from mesenteries and extra vascular tissues. The tissues were mounted on to a tissue bath containing Tyrode's solution maintained at 37 °C and aerated continually with carbogen. The composition of Tyrode's solution (mM) was: KCl 2.7, NaCl 136.9, MgCl2 1.1, NaHCO3 11.9, NaH2PO4 0.4, CaCl2 1.8 and glucose 5.6 (pH 7.4). A preload of 1 g was applied to the tissue and kept undisturbed for 30 min. After stabilization, a sub-maximal dose of acetylcholine (0.3 μM) was used to obtain control responses. The tissue was considered stable when reproducible responses were obtained. Rabbit jejunum produced spontaneous contractions under experimental conditions, so this allows testing spasmolytic or spasmogenic activity of the tissue without the use of an agonist. The effect of essential oil was calculated as percent of the spontaneous contraction or high K+ -induced contractions.

Rabbit jejunum was used to find out the effect of essential oil spontaneous contraction and its mechanism of action. A high concentration of K+  (80 mM) was used for the pre-contraction of the tissue. As plateau, the essential oil was applied in an increasing order of dose to attain the concentration-dependent inhibitory curves. Substances inhibiting high K+ pre-contractions are considered possible Ca++  entry blockers (Karaki & Weiss [15]). For the determination of calcium channel blocking effect, normal Tyrode's solution was replaced with Ca++-free Tyrode's solution having ethylenediamine tetraacetic acid (EDTA, 0.1 mM) for 30 min. Then this solution was also replaced with Ca++-free and K+-rich Tyrode's solution consisting of (mM): KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.87, NaH2PO4 0.41, glucose 5.55 and EDTA 0.1. After 30 min of tissue stabilization, the cumulative CaCl2 concentration response curves were produced in the absence (control) and presence of different concentrations of the essential oil. All changes in isometric tension were recorded through a force transducer coupled with a bridge amplifier data acquisition system (AD Instruments, Sydney, Australia) (Shah et al. [25]).

Wherever applicable, the data were expressed as mean ± standard error of mean (SEM). Two-way ANOVA (Bonferroni post-test) was used for analysis of various parameters of calcium curves. p values less or equal to 0.05 was considered as statistically significant (Graphpad prism software; version 5, GraphPad Software Inc., San Diego, CA).

The GC-MS analysis of the essential oil derived from P. guajava led to the identification of seven major volatile components. The GC chromatogram for these compounds is shown in Figure 1. The retention time (min), molecular weight and molecular formula for each identified compound are listed in Table 1.

Graph: Figure 1. GC chromatogram showing the identified components of essential oil obtained from the fresh fruit of Psidium guajava.

Table 1. Identified components of essential oil obtained from Psidium guajava by the process of steam distillation.

The essential oil of P. guajava produced vasorelaxant effects at doses of 3–10 mg/mL and is shown in Figures 2(b) and 3(a). Figure 2(a) shows the addition of K+  (80 mM) without essential oil. The results showed that essential oil inhibited the PE (1 μM) and K+  (80 mM)-induced sustained contractions, with EC50 values of 6.23 (5.0–7.46) and 5.52 mg/mL (5–6.04), respectively (Figure 3(a)). Verapamil was used as a standard Ca++ channel blocker, which inhibited PE and high K+ pre-contractions with EC50 values of 0.52 (0.3–0.74) and 0. 0.04 μM (0.03–0.05), respectively (Figure 3(b)).

Graph: Figure 2. Typical tracing showing (a) K+  (80 mM) addition without essential oil, (b) the effect of the essential oil from the fresh fruits of Psidium guajava (Pg. Eoil) on K+ (80 mM) and (c) phenylephrine (PE)-induced vasoconstrictions in isolated rabbit aorta preparations.

Graph: Figure 3. Graph shows the concentration-response curve of the essential oil from the fresh fruits of Psidium guajava (Pg. Eoil) (a) and verapamil (b) on K+ (80 mM) and phenylephrine (PE)-induced vasoconstrictions in isolated rabbit aorta preparations. Values shown are mean ± SEM (n = 3).

Psidium guajava essential oil produced a concentration-dependent inhibition of spontaneous (Figure 4(b)) and K+  (80 mM)-induced contractions (Figure 5(a)), with respective EC50 values of 0.84 (0.3–1.38) and 0.71 mg/mL (0.3–1.12), similar to that of verapamil (Figure 5(b)) with an EC50 value of 0.45 (0.3–0.60) and 0.31 μM (0.3–0.32). Figure 4(a) shows normal contraction and relaxation in the rabbit jejunum. The pretreatment of isolated tissues with the essential oil of P. guajava (0.1–1.0 mg/mL; n = 3) shifted calcium curve to right (Figure 6(a)), similar to verapamil (Figure 6(b)), suggesting a spasmolytic effect. The essential oil significantly suppressed and rightward displaced the CaCl2 concentration response curves. This effect was statistically significant (n = 3; *p ≤ 0.05) at concentrations of 0.3 and 1 mg/mL but insignificant at 0.1 mg/mL dose.

Graph: Figure 4. Typical tracing showing (a) normal contraction and relaxation of rabbit jejunum and (b) the concentration-dependent spasmolytic effect of essential oil from fresh fruit of Psidium guajava (Pg. Eoil) on spontaneously contracted isolated rabbit jejunum preparation.

Graph: Figure 5. Graph shows the concentration-dependent inhibitory effect of essential oil from fresh fruit of Psidium guajava (Pg. Eoil) (a) and verapamil (b) on spontaneous and high K+ -induced contractions, in isolated rabbit jejunum preparations. Values shown are mean ± SEM (n = 3).

Graph: Figure 6. The relaxing effect of essential oil derived from Psidium guajava (a) and verapamil (b) showing in the figure on calcium concentration–response curve in isolated rabbit jejunum preparations. Values shown are mean ± SEM (n = 3, *p ≤ 0.05).

Generally, fruits have a beneficial effect on human health. Different fruits like apricot, apple, figs and pomegranate have been reported in the literature for gastrointestinal and cardiovascular diseases (Boyer & Liu [3]; Guarrera [13]; Sharma et al. [29]; Sreekumar et al. [32]). The Guava seeds had considerable effect of lowering the levels of fat, glucose and cholesterol and other risk factors of cardiovascular diseases (Farinazzi-Machado et al. [8]). It has been reported that decoction of the young leaves and shoots of P. guajava are used as spasmolytic in diarrhoea (Shah et al. [26]). From the chemical point of view, the essential oil composition frequently changes in different parts of the plant. Quite often, among the different organs of the plant, phytochemical polymorphism can be produced. As an example, in Origanum vulgare ssp. hirtum, polymorphism could be detected, even within one individual plant, between different oil glands of a single leaf (Johnson et al. [14]). However, this kind of polymorphism is not very usual, being the difference in the oil composition between glands usually related to gland age (Johnson et al. [14]). In general, the different growth stages of the plant create variations in the oil composition within the same part of the plant (Chamorro et al. [6]). It has been previously noted that the essential oil composition may vary greatly depending on the habitat, location, climatic conditions and soil biology (Gholivand et al. [10]). Current literature lacks the pharmacological studies on the essential oil from the fruits of P. guajava in cardiovascular and gut disorders despite its wide spread uses in traditional medicine for these effects. Therefore, this study was carried to investigate the effects of P. guajava essential oil in vascular and gastrointestinal disorders.

The essential oil was analyzed to identify some major chemical constituents. The major chemical constituents identified in the essential oil include butanoic acid methyl ester, 3-methyl glutaric anhydride, 1-butanol, 3-hexenal, cinnamyl alcohol, 1-hexanol and hexene with corresponding retention times of 7.2, 9.5, 10.7, 6.6, 10.9, 13.9 and 7.7 min, respectively. However, few peaks remained unidentified. The compound like 3-hexenal has already been reported in the mature fruit of P. guajava. The compounds 3-hexenal and 2-hexenal imparts flavour of mature Guava fruit (Chyau et al. [7]). Some of the constituents identified in the essential oil of Guava fruit have been reported to possess important biological activities. For example, cinnamyl alcohol has carminative, astringent, antibacterial and stomachic (Lee & Ahn [18]) activities. Further study is needed to explain the monomer composition of the essential oil of Guava and molecular mechanism of the essential oil for gastric and cardiovascular activities.

Based on the medicinal use of Guava fruit in cardiovascular disorders, we tested its essential oil in rabbit isolated aortic rings precontracted with high K+ and phenylephrine. Both high K+  (80 mM) and phenylephrine are known to increase in intracellular calcium through voltage-dependent Ca++ channels (VDCs). The essential oil induced a vasorelaxant effect against both vasoconstrictions, suggesting that it interferes Ca++ entry through VDCs and indicates the presence of Ca++ entry blocking constituent(s). Calcium channel blockers are used clinically for the management of hypertension as vasodilators (Katz [16]). The presence of calcium channel blocking activities in the essential oil provides a pharmacological base to the medicinal use of P. guajava in cardiovascular disorders particularly hypertension.

Guava is considered a useful remedy for the treatment of gastrointestinal disorders, such as diarrhoea, vomiting, gastroenteritis, ulcers and spasms (Ticzon [33]; Lutterodt et al. [19]; Ross [24]; Shen et al. [30]). We wondered if the calcium channel blocking activity of the oil might play role in the management of diarrhoea and/or gut spasms. We tested the oil in isolated rabbit jejunum strips suspended in tissue baths. Rabbit jejunum was allowed contracting spontaneously without using any spasmogenic agent. The essential oil was added cumulatively to see possible inhibitory effect. The essential oil inhibited the spontaneous contractions, suggesting smooth muscle relaxant effect. To investigate the possible mechanism of spasmolytic action, high K+  (80 mM) was applied. High K+ is known to increase intracellular Ca++ through opening VDCs and induces a sustained contraction. Generally, any molecule or chemical inhibiting the K+  (80 mM) produced contractions is recognized as a possible calcium channel-blocking agent (Godfraind et al. [11]). The essential oil induced relaxation of high K+ pre-contractions, suggests that the spasmolytic activity of essential oil acts through calcium channel blockade. Interestingly, the essential oil was about seven times more potent in smooth muscle of intestine than the vascular, indicating tissues selectivity. We further confirmed the calcium channel blocking activity of the essential oil. Pretreatment of the jejunal preparations with essential oil, concentration dependently suppressed the CaCl2 induced concentration response curves, in Ca++ free EDTA solution, similar to that observed with verapamil, a standard calcium channel blocker (Fleckenstein [9]). Generally, calcium channel blockers have a role in the management of diarrhoea and gut spasm (Brunton [4]). These data indicate that the essential oil possesses calcium channel blocking activity, which partly explain the medicinal use of Guava in diarrhoea and gut spasms.

In summary, the data indicate identification of seven new phytoconstituents in the essential oil of P. guajava. Studies in vascular and intestinal smooth muscle preparations indicate the presence of calcium channel blocking activity, which is more selective in the intestinal than vascular tissues and provides sound mechanistic base to its medicinal importance. Further studies are needed to explore the molecular nature of these effects.

The manuscript was reviewed for language/grammar and US English by Christie Ronge, B.S. Chemistry, Department of Pharmacy, Froedtert & The Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, WI. The authors wish to thank Ms. Ronge (christie.ronge@yahoo.com) for her assistance.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was partly carried out with research facilities provided by COMSATS Institute of Information Technology, Abbottabad Campus.